The centromere is essential for chromosome segregation and genome stability. It is the site of kinetochore assembly and chromosome attachment to the spindle microtubules, and it is important for chromosome movement during mitosis and meiosis. Normal human chromosomes have one centromere, but genome rearrangements that occur in malignant or aging cells produce chromosomes with two centromeres, called dicentrics. Barbara McClintock demonstrated nearly 70 years ago that dicentric chromosomes are associated with breakage and instability. However, dicentric chromosomes in humans are unusually stable, presumably due to the poorly understood phenomenon of centromere inactivation. Key centromere and kinetochore proteins are not present at inactive centromeres, but beyond these observations, the process of centromere inactivation is unclear. Epigenetic and sequence-dependent factors are known to contribute to centromere specification, but requirements for centromere assembly, maintenance, and suppression remain obscure. In this proposal we will: 1) determine the mechanism(s) by which de novo dicentric chromosomes are stabilized and 2) establish and test the epigenomic, temporal, and mechanistic basis of centromere inactivation. Proper centromere function at all chromosomes is crucial to organism viability and cell health, emphasizing the need to understand mechanisms driving dicentric formation and centromere inactivation. Dicentric formation is linked to birth defects, infertility, and cancer and occurs with increasing frequency in aging cells. These experiments will improve the current understanding of relationships in genome stability, chromosome biology, and nuclear architecture. The outcomes will provide new information regarding the importance of chromatin requirements in centromere organization and function and identify pathways and components that stabilize structurally abnormal chromosomes. PUBLIC HEALTH RELEVANCE: Dicentric chromosomes are abnormal chromosome rearrangements associated with aging, cancer, birth defects like Down syndrome, miscarriages, and infertility. These chromosomes contain two centromeres instead of only one, and in many organisms, this makes them inherently unstable and prone to breakage. In humans, however, dicentric chromosome rearrangements are common and remarkably stable due to the poorly understood process of centromere inactivation. The objective of this proposal is to understand how and why dicentric chromosomes are so prevalent in humans, particularly as cells age, as well as to dissect the mechanisms of centromere inactivation and chromosome stability.